1. Field of the Invention
This invention relates to a method of detecting nucleic acid hybridization in an electrochemical assay. More particularly, the invention relates to such a method of detecting nucleic acid hybridization in the presence of molecular oxygen. Preferred embodiments include the use of ruthenium amperometry to detect hybridization of DNA or RNA molecules to detection probes immobilized on a detector, preferably a universal chip having gold or carbon electrodes.
2. Description of the Related Art
One method to detect nucleic acid hybridization is to detect a quantity of counterions surrounding the nucleic acid. Accordingly, hybridized nucleic acid would tend to be surrounded by more of the counterions than would single stranded nucleic acid. The counterions are typically detected by an electrochemical reaction, for example by reduction of a trivalent ion to divalent; in this way, the counterions function as an electron transfer species.
Electrochemical quantitation is described in A. B. Steel et al., Electrochemical Quantitation of DNA Immobilized on Gold, Anal. Chem. 70:4670-77 (1998), hereby expressly incorporated by reference in its entirety. In this publication, Steel et al. describe the use of cobalt (III) trisbipyridyl and ruthenium (III) hexaamine as species which interact with surface-immobilized DNA.
The complex Ru(NH3)63+ has a reduction potential on a gold electrode of approximately −250 mV versus Ag/AgCl reference. This potential overlaps with the potential range at which diatomic oxygen (O2) is reduced on a gold electrode at neutral pH. If oxygen is present during an assay using Ru(NH3)63+, reduction of the oxygen causes a background signal that interferes with the interpretation of the current associated with the reduction of Ru(NH3)63+.
One technique used to diminish oxygen's effect is to remove the oxygen in close proximity to the electrochemical cell that would be present in ordinary laboratory conditions. This can be achieved by deaeration with another gas, such as nitrogen or argon. The inert gas is typically administered from a tank to the electrochemical cell before and during the assay to minimize the amount of oxygen present. However, because of the additional steps and equipment involved, deaeration procedures are generally inconvenient, time-consuming, and expensive.
Accordingly, there exists an unmet need in the art for a method of accurately detecting DNA hybridization despite the presence of molecular oxygen in the assay environment.